Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry

Abstract

Since sarcosine and D,L-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine-alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R(2) = 0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of D,L-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb-1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from D,L-alanine leading to underivatized quantification of sarcosine.

A reflectometric interferometric nanosensor for sarcosine

“…In the literature, separation and determination of sarcosine in biological matrix is critical due to its similarity with high‐abundant l ‐alanine. Also, the sarcosine/alanine ratio has been found to be significantly higher in urine derived from biopsy positive PCa patient compared with biopsy‐negative controls . Therefore, the real time alanine detection studies were also performed to show specificity of sarcosine imprinted RIfS nanosensor as given above.…”

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

“…These can be measured by monitoring the changes in the electron transfer properties of well-known redox systems, such as [Fe(CN) 6 ] 4À /[Fe(CN) 6 ] 3À (Fig. 5).…”

“…5 clearly show an increase in the resistance of charge transfer when the several sensor layers are built. This reflects the introduction of negative charges at the electrode surface and the increase in the difficulties in the transport of [Fe (CN) 6 ] 4À /[Fe(CN) 6 ] 3À ions toward the electrode surface after the entrapment of the enzyme (this corresponds to the highest increase in charge transfer resistance). Data was fitted to the Randles equivalent circuit in order to extract the numerical values of the charge transfer resistance and those values are displayed in Table 1.…”

“…2) was placed at the surface of the sensors and a potential scan was applied using a potential range of À1.5 to 0.9 V. EIS assays were made with redox couple [Fe(CN) 6 ] 3À/4À at a potential of +0.12 V, using a sinusoidal potential perturbation with an amplitude of 0.01 V and the number of frequencies equal to 50, logarithmically distributed over a frequency range of 0.1-100 kHz.…”

“…The analytical methods available for direct determination of sarcosine in urine or serum often include chromatography [5][6][7][8][9] and differential mobility analysis [10] with mass spectroscopy. The major disadvantages of these techniques are high instrumentation costs, and complex sample preparation and skilled operator requirements, which is not suitable for routine analysis.…”